WO2004104045A1

WO2004104045A1 - Therapeutical use of anti-cd70 antibody for treating or preventing aids

Info

Publication number: WO2004104045A1
Application number: PCT/NL2003/000385
Authority: WO
Inventors: Marinus Hendricus Jozef Van Oers; Rene Antonius Wilhelmus Van Lier
Original assignee: Stichting Sanquin Bloedvoorziening
Priority date: 2003-05-23
Filing date: 2003-05-23
Publication date: 2004-12-02
Also published as: AU2003237076A1

Abstract

The invertion relates to the therapeutical use of an anti-CD70 antibody for preventing a decrease in the number of T cells i.e. for the treatment or preventing Aids (characterised by T cell immunodeficiency). It is shown that chronic stimulation of murine T cells via CD70 induced excessive effector/memory T cell formation in a CD27- and antigen-dependent but IFN-g-independent manner. At the same time the naive T cell population is progressively depleted from the lymph nodes, which undermines the integrity of the immune system and ultimately leads to host death through opportunistic infection.

Description

THERAPEUTICAL USE OF ANTI-CD70 ANTIBODY FOR TREATING OR PREVENTING AIDS

The invention relates to the field of molecular biology and immunology. More in particular the invention relates to methods for preventing a side-effect of excessive immune activation, even more particular the invention relates to methods for preventing a decrease in the number of T cells in a system that 5 comprises T cells. The invention also relates to a compound capable of decreasing a co-stimulatory signal for T cell expansion. The invention furthermore relates to the treatment of a disease characterised by a decrease in the number of T cells.

0 The outcome of adaptive immune reactions depends on interactions between an abundance of cell surface receptors on lymphocytes and their cognate ligands¹. These receptor-ligand systems not only control the size of the expanded antigen-specific lymphocyte pool but also its effector functions and migratory potential. Receptors fall into distinct protein families, such as the 5 immunoglobuline supergene family and the cytokine receptor superfamily, and signaling outcome is largely dictated by evolutionary conserved signaling domains, located in their cytoplasmic portions, e.g. immunoreceptor tyrosine- based activation motives. Cellular responses following receptor triggering depend on other receptors that are ligated in series and/or in parallel, and the 0 strength and duration of receptor-ligand interactions.

The tumor necrosis factor (TNF) receptor family is a group of type-I transmembrane molecules that share sequence homology in their cysteine- rich, extracellular, ligand-binding domains. Based on their cytoplasmic moieties TNF receptor family members can be divided into two classes. 5 Receptors that contain a death-domain can convey an apoptotic signal², whereas receptors that contain a TNF receptor-associated factor (TRAF)- binding-domain regulate diverse immunological processes including proliferation, survival, effector function and migration³-⁴. Although a number of TNF receptor molecules bind similar TRAF molecules, gene knock-out strategies have revealed unique, non-redundant roles for individual molecules in regulating T cell immunity. OX-40"^A mice and OX-40L⁷" mice are impeded in mounting sufficient CD4⁺ T cell responses, which is reflected in a poor delayed- type-hypersensitivity response⁵-⁶. In contrast, 4-lBB ^/- mice are impaired in CD8⁺T cell responses to viral pathogens⁷ while in CD27⁷- animals both CD4⁺ and CD8⁺ T cell responses are reduced after influenza virus challenge⁸. In part, these differences might originate from the distinct expression patterns of these receptors.

In addition to these loss-of-function mutations, which highlight the biological relevance of TNF receptor family members, several gain-of-function mutations show that receptor signaling needs to be limited to maintain homeostasis within the immune system. In humans, recessive mutations in the extracellular portion of TNF receptor type I have been identified that prevent physiological receptor cleavage. The inability to desensitize this receptor leads to a clinical syndrome with periods of fever and serositis⁹. In mice, targeted expression of TNF to the synovium is sufficient to induce arthritis, whereas transgenic (Tg) expression of TNF in the β cells in the pancreas contributes to the breakdown of tolerance to these cells and finally to diabetes¹⁰. Constitutive expression of BAFF (for B cell activating factor belonging to the TNF family) induces B cell hyper activation and a systemic lupus-like syndrome¹¹.

The CD27 ligand CD70, is an activation molecule that is predominantly expressed on stimulated lymphocytes after antigenic stimulation¹²"¹⁴. Transgenic expression of CD70 on B cells (CD70 Tg) enhances formation of both CD4⁺ and CD8⁺ effector/memory, interferon gamma (IFNγ)-secreting T cells¹⁵.

We now show that chronic activation of the immune system and as a consequence the presence of a hyperactive immune system (or excessive immune activation, the terms will be used interchangeable herein), leads to a depletion of the naϊve T cell compartment and subsequent death from opportunistic infection. These results show that persistent (or chronic or continuous or excessive; the terms may be used interchangeably herein) immune activation per se can result in a state of lethal immunodeficiency.

In (aged) CD70 Tg mice a conspicuous number of phenomena are found that are considered hallmarks of HIV- 1 -induced immunodeficiency: evidence for increased T cell turnover²⁴; initial lymphadenopathy followed by depletion of lymph nodes²⁵; diminution of the naive CD4⁺ and naive CD8⁺ T cell population²⁶; and a progressive inability of T cells to respond ex vivo to antigen and mitogenic stimuli²⁷. The present invention provides an (alternative) method for the treatment of a disease characterised by a progressive decrease in the number of (naive) T cells, wherein said decrease is induced by the presence of a chronic activation of the immune system. For example, the present invention provides a method for the treatment of HIV- 1 infected subjects. In a first embodiment the invention provides a method for at least in part preventing a decrease in the number of T cells in a system that comprises T cells comprising providing said system with a compound capable of decreasing a co-stimulatory signal for T cell expansion.

Adaptive immune responses to pathogens require proper activation, expansion, differentiation and migration of antigen-specific lymphocytes. T cell activation is initiated by ligation of a T cell receptor (TCR) by peptide-MHC complexes. However, proliferation and acquisition of effector functions by naive T cells only ensues when additional or co-stimulatory signals are provided by antigen presenting cells (APC). One example of such a co- stimulatory signal is the receptor CD28 which can bind both to CD80 and CD86 molecules. Other examples of receptors are CD27, OX-40 and 4- IBB.

In a preferred embodiment, said co-stimulatory signal for T cell expansion comprises a CD70-CD27 induced signal. And even more preferably, said co-stimulatory signal for T cell expansion is continuous. As disclosed herein within the experimental part, CD70 transgenic mice show a progressive conversion of naive T cells into effector-memory cells, which culminate in the depletion of naive T cells from lymph nodes and spleen. These T cells changes are depended on continuous CD27-CD70 interactions and T cell antigen receptor stimulation. The presence of a continuous co-stimulatory signal for T cell expansion may for example be obtained as outlined herein within the experimental part. Under normal/natural conditions CD70 is only transiently upregulated on both murine and human T, B and dendritic cells after stimulation. Expression of CD70 appears to be under direct control of antigen since withdrawal of the antigen-receptor signal results in a rapid loss of cell surface expression.

The presence of a "continuous co-stimulatory signal for T cell expansion" results in an immune system that is in essence stimulated for a prolonged period of time (and hence results in a hyperactive immune system). Such a situation is for example obtained in a subject that suffers from a chronic viral infection (for example HIV). In such a subject the presence of a continuous co- stimulatory signal for T cell expansion and as a result a hyperactive immune system will essentially be controlled by the presence of an antigen. However, it is also possible to obtain a "continuous co-stimulatory signal for T cell expansion" irrespective of the presence or absence of an antigen, for example by placing CD70 under control of a constitutive promoter. Said CD70 may for example be expressed on a B cell or a T cell and said promoter is for example a CD 19 or a CD2 promoter.

Hence, said "continuous co- stimulatory signal for T cell expansion", leading to a hyperactive immune system, is essentially persistently present although not necessarily at a constant level, moreover not all immune cells capable of providing a co-stimulatory signal for T cell expansion need to be involved in the initiation or maintenance of said "continuous co-stimulatory signal for T cell expansion". For example, when the level of a chronic active viral infection decreases the co-stimulatory signal for T cell expansion may also decrease. O 2004/104045

The term "at least in part preventing a decrease in the number of T cells" is defined herein as that the number of T cells decline at a slower rate or maintains approximately the same (hence, no further decrease and/or increase in the number of T cells) or that the number of T cells increases due to the application of said method. For determining the fate of the number of T cells preferably a comparison is made to the number of T cells in a control group not treated according to the method of the invention. Preferably, the number of T cells is increased to a level that corresponds to a normal level. For example, the present study with CD70 Tg mouse show that persistent immune activation per se results in a state of lethal immunodeficiency. When the method according to the invention is applied to such a CD 70 Tg mouse the number of T cells is preferably increased to a number/level of T cells present in a mouse which does not show/suffer from a persistent immune activation. Preferably, the invention provides a method for at least in part preventing a decrease in the number of T cells in a system that comprises T cells comprising providing said system with a compound capable of decreasing a co-stimulatory signal for T cell expansion, wherein said T cells are naive T cells. It is disclosed herein within the experimental part that especially the number of naive T cells is reduced as a consequence of the continuous active immune system or as a consequence of constitutive expression of CD70. Naive T cells are typically defined as CD45RA+CD27+CCR7+ cells. Because the pool of other subsets of T cells is influenced by the pool of naive T cells, it is clear that the invention also provides a method for at least in part preventing a decrease in other subsets of T cells. A "system that comprises T cells" may be an in vivo as well as an in vitro system. An example of in vivo system is exemplified herein by a mouse. Said mouse for example expresses CD70 on a B or T-cell and preferably the - expression of CD70 is continuous. Such continuous expression may for example be obtained by operable linking the coding region of CD70 (or a functional fragment and/or equivalent thereof) to a constitutive promoter, for example the CD 19 promoter for continuous expression on B cells or the CD2 promoter for continuous expression on T cells. However, it is clear that (continuous) expression of CD70 may also be induced in other non-human animals, such as a rabbit or a rat. In a preferred embodiment, the invention provides a method for at least in part preventing a decrease in the number of T cells in a system that comprises T cells comprising providing said system with a compound capable of decreasing a co- stimulatory signal for T cell expansion, wherein said continuous co-stimulatory signal for T cell expansion is induced by a chronic active viral infection. Even more preferably, said chronic active viral infection is HIV. Another example of a viral infection is Classical XLP (x-linked lymphoproliferativedisease). Non-classical XLP patients typically die because of a fatal EBV infection. This infection is, just like a HIV infection, manifested by an excessive immune activation and a collapse of the immune system. It is clear from the present disclosure that the observations made in (aged) CD70 Tg mice show a number of phenomena that are considered to be hallmarks of chronic active viral infections. It is also clear from the present application that providing a compound capable of decreasing a co-stimulatory signal for T cell expansion reduces the negative effects (i.e. the reduced number of T cells) induced by the presence of a continuous hyperactive immune system. Hence, such a compound is also applied to at least in part prevent a decrease in the number of T cells in a subject with a chronic active viral infection.

By providing a system that comprises T cells and in which a continuous co-stimulatory signal induces a decrease in the number of (naive) T cells is present, with a compound capable of decreasing a co-stimulatory signal for T cell expansion, negative effects induced by said continuous co-stimulatory signal are at least in part reduced and preferably completely diminished. The number of T cells declines at a slower rate or even maintains at a constant level or increases. And hence, at least one of the detrimental effects of a viral infection, such as HIV, is reduced or preferably completely diminished. It is clear to a person skilled in the art that the compound capable of decreasing a co-stimulatory signal for T cell expansion may be any kind of compound, for example a chemical or synthetic or proteinaceous compound. Preferably, the compound binds to CD70 in such a way that said CD70 cannot bind to CD27 and hence the CD70-CD27 induced signalling is decreased and preferably completely inhibited. In yet another embodiment, the compound binds to CD27 in such a way that (i) CD70 cannot bind to said CD27 and (ii) that said compound binds to CD27 without inducing signalling. Preferably, said compound is a proteinaceous substance. A proteinaceous substance is defined as a substance comprising a peptide, optionally having been modified by for example glycosylation, myristilation, phosphorylation, the addition of lipids, by homologous or heterologous di- or multimerisation, or any other (posttranslational) modifications known in the art. More preferably, said proteinaceous substance is an antibody and even more preferably, said antibody is an anti-CD70 antibody or a functional equivalent and/or a functional fragment thereof. As disclosed herein within the experimental part, a neutralizing CD70 monoclonal antibody (mAb; clone 3B9) reverses detrimental effects of chronic immune activation. A functional equivalent and/or a functional fragment of such an antibody is typically defined as a equivalent and/or fragment that is still capable of neutralizing an effect induced by the presence of a continuous co-stimulatory signal although that might take place at different concentrations/amounts. An example of a functional fragment is a Fab fragment of an antibody and an example of a functional equivalent is an antibody that comprises (non-essential) point mutations. Furthermore functional equivalents are also obtained by producing multiple (monoclonal) antibodies directed against CD70 and then testing whether said obtained (monoclonal) antibodies are able to (functionally) compete with clone 3B9. These kinds of equivalents are obtained by routine experiments and hence are also included herein. In another embodiment, the invention provides a method for obtaining a compound capable of decreasing a co-stimulatory signal for T cell expansion, said method comprising the steps of

- providing a system that comprises a decreasing number of T cells due to the presence of a said co- stimulatory signal with a candidate compound

- determining whether said candidate compound is capable of at least in part preventing a decrease in the number of T cells in said system.

Preferably, said T cells are naive T cells and even more preferably said co-stimulatory signal for T cell expansion comprises a CD70-CD27 induced signal and even more preferably, said co-stimulatory signal for T cell expansion is continuous. Such a system will, dependent on the age of said system, show a decrease in the number of T cells and hence the effect of the addition of a candidate compound to (a certain stage) said system is then used for determining whether said candidate compound is capable of at least in part preventing a decrease in the number of T cells in said system.

As disclosed herein. within the experimental part, continuous expression of CD70 results in combined changes in spleen and PLN that in turn result in a diminution of the naive T cell population in older CD70 Tg animals. Such an animal is very useful for determining whether a compound is capable of decreasing a co-stimulatory signal for T cell expansion. A candidate compound is provided to such an animal and the effect of the candidate compound on the number and/or type of T cells is determined by methods known to the person skilled in the art. When the application of the candidate compound to such an animal at least in part prevents a (further) decrease in the number (and/or) type of T cells, such a compound is then applied to other methods according to the invention. More specific, such a positively identified compound may then be used in other subjects that suffer from a decreased number of (naive) T cells. For example, such a compound is then used in a subject that suffers from a disease that results in a decrease in the number of T cells, such as HIV. Hence, the invention also provides an isolated, recombinant or synthetic compound obtainable according to such a method. As already outlined above, said compound may bind to CD70 and/or CD27. Preferably, said isolated, recombinant or synthetic compound is a proteinaceous substance, for example a (synthetic) peptide. More preferably, said proteinaceous substance is an antibody or a functional equivalent and/or a functional fragment thereof. And even more preferably, said antibody is an anti-CD70 antibody or a functional equivalent and or a functional fragment thereof. However, it is clear that an anti-CD27 antibody may also be used as long as the binding of such an anti- CD27 antibody does not lead to signalling which is comparable to signalling induced by CD70-CD27 interaction.

In another embodiment, the invention provides a nucleic acid encoding a proteinaceous substance according to the invention or a vector comprising such a nucleic acid. Preferably, the invention provides a gene delivery vehicle comprising a vector according to the invention. Vectors and gene delivery vehicle are well known by a person skilled in the art and hence no further . information on this subject matter is provided. With such a vector and/or a gene delivery vehicle a gene that for example encodes (a functional part of) an antibody is used to provide a subject in need of treatment with said (functional part of said) antibody.

In yet another embodiment the invention provides a pharmaceutical composition comprising an isolated, recombinant or synthetic compound or a nucleic acid or a vector according or a gene delivery vehicle according to the invention. A pharmaceutical composition may either be in a solid form (for example, a pill) or in a fluidised form (for example, a liquid formulation). It is clear to a person skilled in the art that the active ingredient (for example, a CD70 mAb) may further be accompanied by a pharmaceutical acceptable carrier or diluent. Herewith the invention provides a method for treating an individual carrying a disease characterised by a decrease in the number of T cells or more preferably a progressive decrease in the number of T cells comprising treating said individual with a pharmaceutical composition according to the invention. Preferably, said T cells are naive T cells. More preferably, said disease is characterised by T cell immunodeficiency and even more preferably, said disease characterised by T cell immunodeficiency is AIDS or classical XLP.

In yet another embodiment, the invention provides a method for obtaining a transgenic non-human animal with a decreased number of T cells comprising the steps of

- providing a non-human animal with a nucleic acid encoding a proteinaceous molecule capable of providing a continuous co-stimulatory signal for T cell expansion to cells of said non-human animal

- aging said non-human animal to a stage wherein said number of T cells are decreasing.

Preferably, said T cells are naive T cells. More preferably, said proteinaceous molecule is CD70 or a functional fragment and/or a functional derivative thereof and even more preferably, said CD 70 or a functional fragment and/or a functional derivative thereof is expressed on B cells or T cells.

In yet another preferred embodiment, said non-human animal is a mouse. Hence, the invention also provides a transgenic non-human animal obtainable according to such a method. Such a transgenic non-human animal is for example very useful for testing the in vivo efficiency of an isolated, recombinant or synthetic compound for its capacity to at least in part prevent a decrease in the number of T cells (see above). Examples of other non-human animals are a rabbit or a rat. Preferably, said T cells are naive T cells. As already outlined above, it is clear that the invention also provides a method for at least in part preventing a decrease in other subsets of T cells.

In the experimental part it is shown that chronic stimulation of murine T cells via CD70 induced excessive effector/memory T cell formation in a CD27- and antigen-dependent but IFN-γ-independent manner. At the same time the naϊve T cell population is progressively depleted from the lymph nodes, which undermines the integrity of the immune system and ultimately leads to host death through opportunistic infection. Without being bound to it, applicant proposes that at least two complementary mechanisms may account for the selective depletion of T cells from the lymph nodes, but not spleen. First, T cell activation initiates a differentiation process in which migratory properties change. Effector/memory T cells loose CCR7, a chemokine receptor that directs homing towards secondary lymphoid organs. CCR7" - T cells have a severely impeded potential to localize in lymph nodes while migration towards the spleen is intact¹⁸. As the majority of T cells in CD70 Tg mice has a effector/memory phenotype, CD44^hiCD62L^ne§, it is likely that these cells home to spleen and other parts of the body but are excluded from lymph nodes. Second, it was found that as CD70 Tg mice age, thymic cellularity sharply drops. Thymic involution appeared to be related to the enhanced antigen- driven effector/memory cell formation since thymocyte numbers were normal in TCR Tg x CD70 Tg mice (numbers in TCR Tg x CD70 Tg mice ranged from 49 to 72 x 10⁶, compared to 3.9 to 9.1 x 10⁶ in CD70 Tg mice). Exhaustion of the naϊve T cell source strongly reduced seeding of these cells to the secondary lymphoid organs and contribute to the involution of the PLN. The reason for thymus dysfunction in CD70 Tg mice is unclear. A direct effect of CD70 on maturation of developing thymocytes is unlikely since only occasional transgenic CD70⁺ B cells could be found in the thymus (data not shown). Also there is no evidence for functional CD27-CD70 interactions in the thymus because CD27 expression on thymocytes, which is down-modulated after CD70 binding, is normal¹⁵. At the time of thymus involution, B cells have virtually disappeared from the secondary lymphoid organs. As this disappearance results from inhibition of B cell development in the bone marrow, we consider it very unlikely that residual mature B cells would preferentially be localized in the thymus. On the other hand we consider the possibility that, in analogy to what has been described in other circumstances, chronic immune activation induces a stress response that through the action of corticosteroids impedes thymic output¹⁹.

Effects of TRAF-binding TNF receptor family members on lymphocyte function are diverse and include increased expansion, acquisition of effector functions and upregulation of chemokine receptors. Several mechanisms may contribute to the observed expansion of effector/memory T cells in CD70 Tg mice. TNF receptor type molecules may act as costimulatory receptors that convert suboptimal signals delivered through TCR-CD3 to mitogenic ones²⁰-²¹. Upregulation of anti-apoptotic molecules such as Bcl-xl and Bcl-2 is induced by OX40 stimulation and enhanced expression of these molecules increases the pool of antigen-primed T cells²². TNF receptor family members, such as 4- IBB, may also increase T cell proliferation²³. It seems clear that these mechanisms are not mutually exclusive and in addition may depend on the strength, duration and context of the specific signal given. In CD27-^Λ animals a decreased antigen-specific T cell pool was found after influenza virus challenge⁸. Costimulation of purified murine T cells in vitro via CD27 is independent of T cell division and suggests that CD27 regulates cell survival⁸. On the other hand, experiments in CD70 Tg mice provided clear evidence for enhanced cell cycle activity, which is in line with the observation that CD70 transfectants induce strong CFSE dilution in anti-CD3 mAb -stimulated naϊve human T cells (not shown). Thus, CD27 signaling may enhance the size of the antigen-stimulated T cell compartment by several mechanisms.

The invention will be explained in more detail in the following detailed description which is not limiting the invention. EXPERIMENTAL PART Materials and Methods

Mice. C57BL/6, CD70 Tg¹⁵, CD27-'-⁸, F5-TCR Tg³⁴, IFNγ-'- (C57BL/6-ifn^¹T^s)³⁵ mice, all on a C57BL/6 background, were bred in the facilities of the

Netherlands Cancer Institute and Sanquin/Research under specific pathogen- free conditions. Identification of mutant mice was performed by PCR analysis of tail DNA or by FACS analysis of peripheral blood cells. All animal experiments were carried out according to institutional and national guidelines and approved by the Experimental Animal Committees of the respective institutes.

Monoclonal antibodies. The following antibodies were obtained from PharMingen (San Diego, CA): CD3ε-allophycocyanin (APC) (clone 145-2C11), CD4-fluorescein isothiocyanate (FITC) or peridinin chlorophyll protein (PerCP) (clone RM4-5), CD8α-phycoerythrin (PE) or PerCP (clone 53-6.7), CD43-FITC (clone 1B11), CD44-PE (clone IM7), CD62L-FITC (clone MEL- 14). The anti- CD3ε (clone 145-2C11), anti-CD70 (clone 3B9) (Tesselaar K, Xiao Y, Arens R, van Schijndel GM, Schuurhuis DH, Mebius RE, Borst J, van Lier RA. Expression of the murine CD27 ligand CD70 in vitro and in vivo. J Immunol. 2003 Jan l;170(l):33-40) were purified from hybridoma culture supernatant following standard procedures.

Flow cytometry. Single cell suspensions were obtained from spleen, lymph nodes (axillary, brachial, and inguinal or mesenteric), and thymus by grinding the tissues through nylon sieves. Cells (4 x 10⁵) were collected in 96-well U- bottomed plates in FACS staining buffer (PBS, 0.5% bovine serum albumin). All samples were preincubated for 10 min with anti-CD16/CD32 (FcBlock, clone 2.4G2, PharMingen) and subsequently stained for 30 min at 4°C with antibodies. After cell-surface staining, intracellular BrdU and Ki-67 staining was performed following the manufacturer's instruction using a BrdU-FITC Flow kit and a Ki-67-PE set, respectively (PharMingen, San Diego, CA). Annexin-V and PI staining was performed with an APOPTEST-FITC kit (Nexins Research BV, Kattendijk, The Netherlands). FACS analysis was performed on a FACSCalibur™ using Cell Quest software (Becton Dickinson).

BrdU labelling. Four-week-old mice were given drinking water containing BrdU (0.8 mg/ml) (Sigma, St. Louis, MO) for 10 days. Mice were sacrificed at 5.5, 6.5, 8.5 and 10.5 weeks of age (i.e. 0, 1, 3 and 5 weeks after stopping BrdU administration) .

Proliferation assay. T cells were purified from mesenteric lymph nodes by negative depletion using rat-anti-MHC-class II, rat-anti-B220 antibodies, goat- anti-rat IgG microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and MACS (BS) depletion columns following the manufacturers instructions.

Purified T cells (> 90% CD3⁺, as analysed by flow cytometry) were cultured in IMDM supplemented with 10% FCS, and stimulated with PHA (1 μg/ml, 10⁵ cells/well) ( Murex, Lenexa, KS) or anti-CD3_ε mAb (clone 145-2C11, immobilised, 10 μg/ml)). [³H]thymidine (0.4 μCi; Amersham International) was added for the last 16 h of a 96-h culture period.

Immunohistochemistry. Hematoxylin/eosin staining and Grocott staining were performed on formalin fixed lung tissue sections following standard procedures.

Antibody treatment. Mice were given anti-mCD70 (3B9) or control hamster mAb (250 μg/ injection, intraperitoneal) twice weekly for a period of 3 weeks.

Influenza virus infection. F5-TCR Tg and F5-TCR Tg x CD70 Tg were anesthetized and infected intranasally with 25 HAU of influenza virus strain A/NT/60/68 for infection. Before (day 0) and at day 9 after infection, blood from tail was collected, erythrocytes were lysed and cells were analyzed by flowcytometry.

EXPERIMENTAL PART Results

Enhanced T cell proliferation in CD70 Tg mice. Transgenic expression of CD70 on B cells (CD70 Tg) enhances formation of both CD4⁺ and CD8⁺ effector/memory, interferon gamma (IFNγ)-secreting T cells¹⁵. Signaling via TRAF-binding TNF receptor family molecules can affect T cell functions through several mechanisms including lowering of thresholds for signals generated by mitogenic receptors such as T cell antigen receptor (TCR)- CD3, enhancing cell cycle activity, and inhibiting apoptosis. To address the mechanism by which transgenic CD70 expression enhances effector T cell numbers, in vivo BrdU pulse-chase labeling experiments were performed. After a 10 day period on BrdU-containing water, CD70 Tg mice showed an approximately 3-fold increase in the percentage of BrdU-labeled CD3⁺ T cells both in peripheral lymph nodes (PLN), and spleen as compared to wild-type (WT) (Fig. la). The percentages of labeled cells rapidly dropped when BrdU feeding was stopped which suggested that the increased labeling was not due to impaired apoptosis, but rather reflected increased cell division.

To analyze this directly we measured the percentages of cycling and apoptotic cells within the T cell pool of PLN and spleen. As measured by Ki-67 expression, CD70 Tg mice had increased percentages of T cells in cycle (Fig. lb) in PLN (3.3% in Tg versus. 1.3% in WT) and spleen (14.5% in Tg versus. 3.3% in WT). FACS analysis of Annexin V and propidium iodide (PI) staining was performed to estimate the percentage of apoptotic cells (Annexin V⁺ PT^or+). T cell death rates appeared to be relatively constant in WT mice and no differences were observed with CD70 Tg mice of 4, 8 and 13 weeks of age (data not shown). In 20-week-old CD70 Tg mice, a small increase in percentage of apoptotic cells was seen in PLN (40% in Tg versus. 27% in WT, p<0.05 student's t-test) and the spleen (42% in Tg versus 23% in WT, p<0.05 student's t-test, data not shown). The ability of T cells to undergo clonal expansion was measured (Fig. lc). Irrespective of age, lymph node T cells of CD70 Tg mice had a reduced ability to divide upon stimulation with anti-CD3 mAb in vitro (reduction in mean [³H]thymidine incorporation of 1.5 to 2.1 fold compared to WT). In contrast, proliferation in response to phytohemagglutinin was normal in 4 week old mice mean [³H]thymidine incorporation of 1.8 fold compared to WT) but dropped as the animals aged (at 20 weeks, reduction in mean [³H]thymidine incorporation of 1.6 fold compared to WT). Thus, T cells from CD70 Tg mice showed increased proliferation in vivo but had a decreased ability to divide upon polyclonal stimulation in vitro.

Diminution of naive T cells in CD 70 Tg mice

To investigate the long-term effects consequences of the enhanced T cell proliferation in vivo, T cell numbers in lymphoid organs of CD 70 Tg mice were analyzed longitudinally. T cell numbers in PLN were elevated in transgenic mice of 4 and 8 weeks of age compared to WT (Fig 2a). Both CD4⁺ and CD8+ T cells contributed to this increase¹⁵ (data not shown). As mice aged, lymph node T cell numbers dropped sharply. At 20 weeks, PLN had strongly involuted and T cell numbers in CD70 Tg mice were below 10% of the amount found in WT mice (Fig. 2a). In contrast to the T cell numbers in PLN, splenic T cell numbers were increased compared to WT mice irrespective of age (Fig 2b). However, accumulation of effector/memory cells appeared to be progressive and at 20 weeks of age the vast majority of splenic T cells, both CD4⁺ and CD8⁺, was of the effector/memory CD44^hiCD62L^nes phenotype (Fig. 2d). The combined changes in spleen and PLN resulted in an almost complete diminution of the naϊve T cell population in older CD70 Tg animals. The CD4/CD8 ratio was not significantly different between CD70 Tg and WT mice, neither in young nor in aged mice (data shown).

Thymocyte numbers were similar in Tg and WT mice until 13 weeks of age, however at 20 weeks of age only thymic remnants with low cellularity could be recovered from the Tg animals (Fig. 2c). Thymocyte differentiation as judged by CD4 and CD8 expression was not changed in CD70 Tg mice (data not shown). Thus, constitutive expression of CD70 did not only result in reduced naϊve T cell numbers in the secundary lymphoid organs but also in diminished production of naϊve T cells in aged mice.

Requirements for effector/memory cell formation.

In contrast to a number of other ligands of the TNF superfamily such as TNF, TRAIL (TNF-related apoptosis inducing ligand) and LIGHT, CD70 appears to have only one receptor, CD27. Indeed, the excessive formation of effector/memory T cells in CD70 Tg mice was completely abolished when these mice were crossed with CD27- - animals (Fig. 3a). This finding is in full agreement with the observation that the T cell and IFNγ-dependent B cell depletion in CD70 Tg mice, can be rescued on a CD27 ^/- background¹⁵. Moreover, expansion of effector/memory T cells was dependent on continuous CD27-CD70 interaction, since treatment of 4-week-old CD70 Tg mice with blocking CD70 mAb reduced the number of effector/memory cells to WT numbers (Fig. 3b). These results demonstrated that the mechanism of the enhanced effector/memory T cell formation was not only initiated but subsequently also maintained by CD27-CD70 interaction.

To test whether the interaction between CD27 and CD70 in the absence of a signal delivered through the TCR sufficed to expand the effector/memory pool, CD70 Tg mice were crossed with mice that carry a major histocompatibility complex (MHC)-class-I restricted transgenic TCR specific for influenza A virus (TCR Tg), which is not present in the mouse colony. In these double transgenic mice CD70 expression did not enhance CD8⁺ effector/memory T cell formation. The CD8⁺ T cell compartments of both TCR Tg and TCR Tg x CD70 Tg mice (which are largely composed of TCR Tg T cells) comprise naϊve T cells (Fig. 4a and b). In contrast, the non-transgenic CD4⁺ T cell compartment in the TCR Tg x CD70 Tg mice predominantly contained effector/memory T cells, as evidenced by expression of the effector T cell marker CD43¹⁶ (Fig. 4b). After intranasal infection with influenza virus TCR Tg x CD 70 Tg mice made effector/memory T cells much more efficiently than single TCR Tg mice (Fig. 4c), corroborating that the effects of persistent CD27-CD70 interactions on T cells requires adtivation of the TCR by antigen. In CD70 Tg mice these signals were likely provided by environmental antigens.

CD70 Tg mice have increased numbers of IFN-γ-secreting T cells and the in vivo effects of this regulatory cytokine are reflected by enhanced MHC class II expression and inhibitory action on B cell precursor cells¹⁵. IFN-γ may also play a role in selection and maintenance of the effector/memory T cell pool¹⁷. Therefore, to test whether IFN-γ has a regulatory role on effector/memory T cell formation in the CD70 Tg mice we analyzed subset distribution and T cell cycling in vivo of 20-week-old IFN-γ- ^_χ CD70 Tg mice. In these mice we also observed a strong skewing towards effector/memory T cells (Fig. 5a) and an increase in the percentage of cycling cells as evidenced by Ki-67 staining (Fig. 5b) similar to findings in IFN-γ-proficient CD70 Tg animals (Fig lb). Thus, excessive formation of effector/memory cells in CD70 Tg mice was dependent on CD27-CD70 interactions and foreign antigens, but independent of IFN-γ.

Premature death of CD70 Tg mice.

During the first months of life CD70 Tg mice appeared healthy, showed enhanced delayed type hyper sensitivity reactions and mounted normal primary antibody responses to protein antigens (Tesselaar K, Xiao Y, Arens R, van Schijndel GM, Schuurhuis DH, Mebius RE, Borst J, van Lier RA.

Expression of the murine CD27 ligand CD70 in vitro and in vivo. J Immunol. 2003 Jan l;170(l):33-40). However, as CD70 Tg mice aged they failed to thrive, resulting at 20 weeks in a body mass of approximately 80% of that of WT animals (Fig. 6a). Most 20-week-old CD70 Tg mice suffered from Pneumocystis carinii pneumonia (Fig. 6b), an opportunistic infection usually seen in situations of severe T cell immunodeficiency,⁷ accompanied by a premature death at an average age of 28 weeks (Fig. 6c). This inability to cope with opportunistic pathogens appeared to be a direct result of the enhanced effector/memory cell formation and the concurrent collapse of the naϊve T cell compartment because Pneumocystis carinii pneumonia was not observed in CD27-^/- x CD70 Tg nor in TCR Tg x CD70 Tg mice (data not shown). IFN-γ-^/- x CD70 Tg animals also suffered from infection resulting in premature death at average age of 30 weeks (Fig. 6c). Thus, premature death in CD70 Tg mice appeared to be directly related to the demise of the naive T cell compartment and was caused by opportunistic infections.

Expression of CD70 on T cells

Experiments on CD 70 Tg mice in which CD 70 was expressed on T cells (under control of the CD2 promoter) resulted in similar phenotypes.

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Figure 1. T cell proliferation in CD70 Tg mice. Flow cytometry was performed to measure (a) T cell expansion and (b) T cell division. Four week old mice were given BrdU in their drinking water for 10 days and the percentage of BrdU⁺ and Ki-67⁺ cells within the CD3⁺ population from spleen (primary axis) or PLN (secondary axis) derived from WT (left panels) or CD70 Tg (right panels) mice sacrificed at the indicated age was determined. Each symbol represents data from an individual mouse; lines represent the mean value of 2 to 6 mice, (c) Proliferative responses of T cells from CD70 Tg mice. T cells were purified from mesenteric lymph nodes derived from WT or CD70 Tg mice at 4, 8 and 13 weeks of age and stimulated with PHA (closed circles) or anti-CD3ε mAb (open circles). The stimulation index is defined as the mean [³H]thymidine incorporation of 3 Tg mice divided by the mean [³H]thymidine incorporation of 3 WT mice. The graph shows the mean value of two experiments +/- standard error of the mean ([³H]thymidine incorporation range: 1396-10219 cpm (PHA stimulation) and 1975-32966 cp (anti-CD3 mAb stimulation)).

Figure 2. T cell cellularity and phenotype in CD70 Tg mice. Absolute numbers of CD3⁺ T cells in (a) PLN and (b) spleen in WT (white) and CD70 Tg (black) mice of 4, 8, 13, and 20 weeks of age were determined. T cell numbers were calculated by multiplying the number of mononuclear cells with the percentage of CD3⁺ T cells as determined by flow cytometry. (c) Thymic cellularity was determined by counting lymphocyte numbers of WT and CD70 Tg mice at the indicated ages. Shown are the mean values of 4 to 6 mice +/- the standard deviation. Differences between mean values of WT and CD70 Tg at the indicated age are considered significant (*) if p<0.05 (Student's t-test). (d) Phenotype of CD4⁺ and CD8⁺ splenic T cells determined by flow cytometry. Naive T cells are being defined as CD44^neε^dullCD62L^hi; effector/memory cells as CD44^hiCD62L^neε. Figure 3. Requirements for enhanced T cell expansion and differentiation in CD70 Tg mice, (a) Dependence on CD27-CD70 interactions. Phenotype of splenic CD3⁺CD8⁺ cells derived from 20-week-old WT, CD70 Tg (F13) and CD27-^/- x CD70 Tg mice was determined by flow cytometry. Indicated are the percentages of naive (CD44^neε^/duUCD62L^hi) and effector/memory (CD44^hiCD62L^ne&) T cells, (b) Dependence on persistent CD27- CD70 interactions. Four-week-old CD70 Tg mice were treated for 3 weeks with control hamster mAb or hamster-anti-CD70 mAb (3B9). Absolute numbers of splenic naϊve, effector/memory T cells were determined at the end of the treatment. T cell numbers were calculated by multiplying the number of mononuclear cells with the percentage of the indicated populations as determined by flow cytometry. Shown are the mean values +/- the standard deviation (n =3).

Figure 4. Effector T cell phenotype of CD70 Tg mice is dependent on stimulation of the TCR by antigen, (a) Representative FACS profiles of splenic CD3⁺CD8⁺ cells derived from 16-week-old WT, CD70 Tg, (MHC-class I restricted) F5 TCR Tg and F5 TCR Tg x CD70 Tg mice were determined by flow cytometry. Indicated are the percentages of naive (CD44^neε^/dullCD62L^hi) and effector/memory (CD44^hiCD62L^nes) T cells, (b) Percentage of CD43 (1B11) positive cells within the splenic CD4⁺ (white) and CD8⁺ (black) population of WT, CD70 Tg, TCR Tg and TCR Tg x CD70 Tg mice, (c) Representative histogram plots showing CD43 (IB 11) staining of CD8+ lymphocytes in blood of TCR Tg and TCR Tg x CD70 Tg mice at day 0 and day 9 after intranasal infection with influenza A virus. Numbers indicate the percentages of CD43⁺ cells within the CD8⁺ population. One representative experiment from three independent experiments is shown.

Figure 5. T cell characteristics of IFNγ-^/- x CD70 Tg mice, (a) Flow cytometric analysis of splenic CD4⁺ and CD8⁺ cells from 20-week-old WT and CD70 Tg. Indicated are the percentages of naive and effector/memory T cells, (b) Flow cytometry was performed to determine the percentage of Ki-67⁺ within the CD3⁺ cell population from spleen and PLN derived from 20-week- old IFNγ-^/- and IFNγ-'- x CD70 Tg mice.

Figure 6. Clinical symptoms in CD 70 Tg mice, (a) Cachexia in CD 70 Tg mice. Male WT and CD70 Tg mice were weighed at 4, 8, 13 and 20 weeks of age. The graph shows the mean weight +/- standard deviation (ri≥6). Significant differences (Student's t-test, p<0.05) between mean values of WT and CD70 Tg are denoted by * (b) Pneumocystis carinii pneumonia in CD70 Tg mice. Hematoxylin/eosin staining (upper panels), and Grocott staining (lower panels) were performed on formalin fixed lung tissue sections derived from WT (left panels) and CD 70 Tg (right panels) mice. Grocott staining shows cysts of Pneumocystis carinii in sections derived from CD 70 Tg mice. Magnification: 62.5 fold, (c) Shortened life span of CD70 Tg mice. Cumulative survival curves for WT, CD70 Tg, IFNγ-'- and IFNy'- x CD70 Tg mice.

Claims

1. A method for at least in part preventing a decrease in the number of T cells in a system that comprises T cells comprising providing said system with a compound capable of decreasing a co-stimulatory signal for T cell expansion.

2. A method according to claim 1, wherein said T cells are naive T cells.

3. A method according to claim 1 or 2, wherein said co-stimulatory signal for T cell expansion comprises a CD70-CD27 induced signal.

4. A method according to any one of claims 1 to 3, wherein said costimulatory signal for T cell expansion is continuous.

5. A method according to claim 4, wherein said continuous co-stimulatory signal for T cell expansion is induced by a chronic active viral infection.

6. A method according to claim 5, wherein said chronic active viral infection is HIV.

7. A method according to any one of claims 1 to 6, wherein said compound is a proteinaceous substance.

8. A method according to claim 6, wherein said proteinaceous substance is an antibody.

9. A method according to claim 8, wherein said antibody is an anti-CD70 antibody or a functional equivalent and/or a functional fragment thereof.

10. A method for obtaining a compound capable of decreasing a co- stimulatory signal for T cell expansion, said method comprising the steps of

- providing a system that comprises a decreasing number of T cells due to the presence of a said co-stimulatory signal with a candidate compound

11. A method according to claim 10, wherein said T cells are naive T cells.

12. A method according to claim 10 or 11, wherein said co-stimulatory signal for T cell expansion comprises a CD70-CD27 induced signal.

13. A method according to any one of claims 10 to 12, wherein said costimulatory signal for T cell expansion is continuous.

14. An isolated, recombinant or synthetic compound obtainable according to the method of any one of claims 10 to 13.

15. An isolated, recombinant or synthetic compound according to claim 14, wherein said compound is a proteinaceous substance.

16. An isolated, recombinant or synthetic compound according to claim 15, wherein said proteinaceous substance is an antibody or a functional equivalent and/or a functional fragment thereof.

17. An isolated, recombinant or synthetic compound according to claim 16, wherein said antibody is an anti-CD70 antibody or a functional equivalent and/or a functional fragment thereof.

18. A nucleic acid encoding a proteinaceous substance according to any one of claims 15 to 17.

19. A vector comprising a nucleic acid according to claim 18.

20. A gene delivery vehicle comprising a vector according to claims 19.

21. A pharmaceutical composition comprising an isolated, recombinant or synthetic compound according to anyone of claims 14 to 17, a nucleic acid according to claim 18, a vector according to claim 19 or a gene delivery vehicle according to claim 20.

22. Use of an isolated, recombinant or synthetic compound according to anyone of claims 14 to 17, a nucleic acid according to claim 18, a vector according to claim 19 or a gene delivery vehicle according to claim 20 for the preparation of a medicament for the treatment of a disease characterised by a decrease in the number of T cells.

23. Use according to claim 22, wherein said T cells are naive T cells.

24. Use according to claim 22 or 23, wherein said decrease in the number of T cells is a progressive decrease in the number of T cells.

25. Use according to any one of claims 22 to 24, wherein said disease is characterised by T cell immunodeficiency.

26. Use according to claim 25, wherein said disease characterised by T cell immunodeficiency is AIDS.

27. A method for obtaining a transgenic non-human animal with a decreased number of T cells comprising the steps of - providing a non-human animal with a nucleic acid encoding a proteinaceous molecule capable of providing a continuous co-stimulatory signal for T cell expansion to cells of said non- human animal

28. A method according to claim 27, wherein said T cells are naive T cells.

29. A method according to claim 27 or 28, wherein said proteinaceous molecule is CD70 or a functional fragment and/or a functional derivative thereof.

30. A method according to claim 29, wherein said CD70 or a functional fragment and/or a functional derivative thereof is expressed on B cells.

31. A method according to anyone of claims 27 to 30, wherein said non- human animal is a mouse.

32. A transgenic non-human animal obtainable according to the method of any one of claims 27 to 31.

33. Use of a non-human animal according to claim 32 for testing the in vivo efficiency of an isolated, recombinant or synthetic compound for its capacity to at least in part prevent a decrease in the number of T cells.

34. Use according to claim 33, wherein said T cells are naive T cells.